Synthesis, characterization, and antibacterial properties of a hydroxyapatite adhesive block copolymer

Article abstract of DOI:10.1021/ma501997x

A novel diblock copolymer composed of bisphosphonate and pyridine oligomers has been prepared by reversible addition-fragmentation transfer (RAFT) polymerization. Ag ion was introduced into the polymer via its coordination with the pyridine groups, followed by a reduction process to obtain Ag nanoparticles with diameters of 5-15 nm measured by transmission electron microscopy (TEM). In addition, X-ray photoelectron spectroscopy (XPS) and X-ray diffraction (XRD) proved successful introduction of Ag nanoparticles into polymer. Ag nanoparticles containing polymer exhibited excellent antibacterial properties toward Lactobacillus plantarum (L. plantarum). In order to investigate its practical application as an antibacterial coating, the synthesized polymer was tethered onto hydroxyapatite (HA, main mineral component of natural bone, teeth, and most of implants for bone repair) surfaces via interaction between the polymers bisphosphonate group and HA, forming ～4 nm thick layers. Ag nanoparticles (5-15 nm in diameter) uniformly distributed around the HA particles were fabricated following the above process. The ability of the coating to kill the bacteria L. plantarum was determined, which revealed strong antibacterial properties.

Full text of DOI:10.1021/ma501997x

Article
pubs.acs.org/Macromolecules
Synthesis, Characterization, and Antibacterial Properties of a
Hydroxyapatite Adhesive Block Copolymer
Qiang Matthew Zhang and Michael J. Serpe*
Department of Chemistry, University of Alberta, Edmonton, AB T6G 2G2, Canada
ABSTRACT: A novel diblock copolymer composed of bisphosphonate
and pyridine oligomers has been prepared by reversible addition−
fragmentation transfer (RAFT) polymerization. Ag ion was introduced
into the polymer via its coordination with the pyridine groups, followed by
a reduction process to obtain Ag nanoparticles with diameters of 5−15 nm
measured by transmission electron microscopy (TEM). In addition, X-ray
photoelectron spectroscopy (XPS) and X-ray diffraction (XRD) proved
successful introduction of Ag nanoparticles into polymer. Ag nanoparticles
containing polymer exhibited excellent antibacterial properties toward
Lactobacillus plantarum (L. plantarum). In order to investigate its practical
application as an antibacterial coating, the synthesized polymer was
tethered onto hydroxyapatite (HA, main mineral component of natural
bone, teeth, and most of implants for bone repair) surfaces via interaction
between the polymer’s bisphosphonate group and HA, forming ∼4 nm thick layers. Ag nanoparticles (5−15 nm in diameter)
uniformly distributed around the HA particles were fabricated following the above process. The ability of the coating to kill the
bacteria L. plantarum was determined, which revealed strong antibacterial properties.
9
10
10a,11
INTRODUCTION
agents, hydrogels, Ag nanoparticles or compounds,
or
■
1
2
various polymers.
Ag is an extremely potent antimicrobial agent that exhibits
strong cytotoxicity toward a broad range of microorganisms,
while exhibiting low known toxicity in humans. Ag exhibits
an oligodynamic effect, that is, Ag is capable of causing a
bacteriostatic (growth inhibition), or even a bactericidal
antibacterial) effect.
that Ag nanoparticles exhibited more efficient antibacterial
performance compared with their bulk counterparts.
Although the debate concerning the mechanisms by which
Ag nanoparticles exert their antibacterial action is still
underway, it is generally accepted that their mechanism of
action involves a release of Ag ions, which then interacts with
and kills bacteria. Recently, a hypothesis has been formulated,
suggesting that the antibacterial properties of AgNPs can be
ascribed to a short distance nanomechanical action involving
The prevention of bacterial adhesion to surfaces is significant to
maintain the function and fidelity of many materials and
processes; most importantly, it benefits human health. Once
adhered onto a solid surface, bacteria form colonies and
subsequent biofilms that serve as reservoirs for the develop-
ment of pathogenic infections such as tooth decay and
biofouling of medical implants and biomedical devices
1
11a
11a
(
Recently, researchers have reported
2
ultimately leading to implant rejection. Implant-associated
13
infections are among the most serious postsurgical complica-
tions from medical device implantation, including prosthetic
joints (e.g., hip, knee, and shoulder) and fracture fixation
3
hardware. Infection rates related to orthopedic implants have
been reduced to less than 5% owing to strict hygienic protocols
and intraoperative systemic prophylactic treatment. However,
4
the overall number of such infections has been continuously
increasing with the growing demand for surgical implantation
as a result of population aging and increasing participation in
14
their direct interaction with the bacterial cell membrane.
Thus, it is very promising to introduce Ag nanoparticles on a
surface for broad antibacterial applications. However, aggrega-
tion of Ag nanoparticles and promoted adhesion of bacteria act
as barriers to fabricating surface coatings that exhibit strong
antibacterial effects as well as biocompatibility using only Ag
nanoparticles. Polymeric materials with great structure
tailorability and flexibility have the potential to inhibit
5
recreational activities. Prevention of biofilm formation has
therefore been of recent interest to biomaterials researchers,
whose efforts have focused on the design of coatings tailored
6
for this purpose. Coating strategies can be classified as passive
7
15
(
antifouling) and active (antimicrobial). Passive strategies rely
on inhibition of bacterial attachment, typically through physical
prevention of nonspecific cell attachment by a grafted polymer
coating, whereas active strategies rely on the presence of an
antibacterial compound that actively promotes bacteria death
by interfering with biochemical pathways. Various surface
modifications have been developed to improve the antibacterial
aggregation of Ag nanoparticles and form uniform surface
coatings on various substrates. These materials can also
16
8
Received: September 26, 2014
Revised: October 28, 2014
properties of implant surfaces, such as applying bactericidal
©
XXXX American Chemical Society
A
dx.doi.org/10.1021/ma501997x | Macromolecules XXXX, XXX, XXX−XXX

Macromolecules
Article
control the release of Ag ions for sustained antibacterial effects
and reduce cytotoxicity.
Na
2
SO
4
, and then the solvents were removed by rotary evaporation.
The resulting oil was purified by column chromatography. Yield: 52%.
1
H NMR (CDCl , ppm): δ 1.36 (t, 12H, −CH ), 3.58 (t, 1H, P−CH−
Hydroxyapatite (HA, [Ca (PO ) (OH) ]) is a bioactive
3
3
1
0
4 6
2
P), 4.07 (m, 4H, N−CH −Ph), 4.21 (m, 8H, O−CH −), 7.20−7.45
(
506.5, obsd 506.5 (M + Na ).
calcium phosphate ceramic that is the main mineral component
2
2
31
m, 10H, Ar−H). P NMR (CDCl , 300 MHz): δ 20.7 (s). MS: calcd
3
of natural bone, teeth, and most of implants for bone repair or
+
1
7
regeneration. Generation of biofunctional surfaces on
hydroxyapatite that inhibit bacterial growth is one of the best
methods to prevent colonies and biofilms on these implants.
However, compared with polymeric and noble metal surfaces, a
hydroxyapatite surface is difficult to modify by graft polymer-
ization or complexation reaction due to lack of functional
groups on the surface. It has been reported that bi-
sphosphonates, structural analogues of naturally existing
pyrophosphate with increased chemical and enzymatic stability,
Synthesis of Tetraethyl Aminomethylbis(phosphonate). A
50 mL three-necked round-bottom flask was flushed with N and
2
2
charged with 10% Pd/C (0.5 g), and a solution of TEBAP (5.0 g, 10.4
mmol) in EtOH (150 mL) was added. The mixture was refluxed with
vigorous stirring under a H atmosphere for 24 h. After filtration and
evaporation of volatiles, the product was obtained as colorless liquid.
2
1
Yield: 93%. H NMR (CDCl , ppm): δ 1.36 (t, 12H, −CH ), 3.43 (t,
3
3
31
1H), 4.23 (m, 8H, O−CH
−). P NMR (CDCl
, ppm): δ 20.8 (s).
2
3
+
MS: calcd 326.2, obsd 326.2 (M + Na ).
18
Synthesis of Monomer (AABP). To a solution of tetraethyl
aminomethylbis(phosphonate) (0.606 g, 2.00 mmol) and triethyl-
amine (0.42 mL, 3.00 mmol) in anhydrous dichloromethane (5.60
mL) was added acryloyl chloride (0.32 mL, 3.30 mmol) in anhydrous
dichloromethane (3.26 mL) at 0 °C under N , and the resulting
mixture was stirred at room temperature for 2 h. The reaction was
stopped by addition of distilled water (3.26 mL). The organic layer
have strong affinity for hydroxyapatite. Moreover, it was also
reported that bisphosphonates can inhibit enzymes (metal-
19
loproteinases), which degrade the collagen network of bone.
In the present paper, we report on the synthesis of a block
copolymer composed of bisphosphonate (block 1) and pyridine
2
(block 2). Bisphosphonate oligomers are hypothesized to tether
the copolymer on the HA surface. Ag can then be incorporated
into this system due to strong coordination capability of
pyridine oligomers. We describe as being scorpion-likethe
bisphosphonate-containing block acts like the scorpion’s legs,
securing the polymer to the surface, while the pyridine/Ag
block acts like the scorpion’s tail, which can kill bacteria. After
careful structural analysis and polymer characterization, the
antibacterial properties of the polymer−Ag and the HA surface
modified by the polymer−Ag were studied. Lactobacillus
plantarum (L. plantarum) was selected as model bacteria,
was washed with distilled H O (25 mL), 2 M HCl (25 mL), saturated
2
NaHCO3 (25 mL), and distilled H O (25 mL) and dried over
2
anhydrous Na SO and filtered. After removal of the solvent, the crude
2
4
product was purified by reversed-phase flash chromatography on silica,
eluting with H O:MeOH (50:50) to give monomer as a white solid.
2
1
Yield: 48%. H NMR (CDCl , ppm): δ 1.30−1.36 (m, 12 H, −CH ),
3
3
4
1
.2 (m, 8 H, P−O−CH −), 5.10−5.15 (d, 1H, CH−P), 5.15−5.19 (q,
2
31
H, CH), 5.71−5.74, 5.30 (q, 2H, CH ). P NMR (CDCl ,
2
3
+
ppm): 16.11 (s). MS calcd 348.1, obsd 348.1 (M + Na ).
Synthesis of Bisphosphonate Oligomers (P(BP)). The polymer
was denoted as p(BP). To a 10 mL round-bottom flask, 0.97 g of
AABP (2.73 mmol), 0.0030 g of AIBN (1:5 molar ratio to RAFT-
COOH), 0.0328 g of RAFT-COOH (0.091 mmol), and 2 mL of DMF
were added and stirred at room temperature until they were
completely dissolved. The solution was sparged with nitrogen gas
for 20 min and then heated to 65 °C for 4 h. The reaction was
terminated by quenching in an ice−water bath to reduce the
temperature. The reaction solution was dialyzed against DI water for
20
which are prominent in decaying plant material and teeth.
EXPERIMENTAL SECTION
■
Materials. 2,2′-Azobis(2-methylpropionitrile) (AIBN) was purified
by recrystallization in methanol. L. plantarum was purchased from
Custom Probiotics Inc. (Glendale, CA). All other chemicals were
purchased from Sigma-Aldrich and used as received.
3
days to remove solvent and the unreacted monomer. The yellow
Synthesis of 2-(Dodecylthiocarbonothioylthio)-2-methyl-
propanoic Acid (RAFT-COOH). 1-Dodecanethiol (8.076 g, 0.040
mol), acetone (19.2 g, 0.331 mol), and Aliquot 336 (tricaprylylme-
thylammonium chloride, 0.649 g, 0.0016 mol) were mixed in a 250 mL
1
solid was dried in vacuo for 12 h. H NMR (DCCl , ppm): 2.98 (4H,
3
m, N−CH and S−CH ), 2.83 (6 H, N−CH ), 2.60 (2 H, S−CH ),
2
2
3
2
2
.12 (2H, m, CH ), 1.76 (2 H, m, CH ), 1.24 (34 H, m, CH ), 0.85
2 2 2
(3H, t, CH3).
flask cooled to 10 °C under N . Sodium hydroxide solution (50%)
2
Synthesis of p(BP-b-VP) Block Copolymer. p(BP) (5 kDa
(
3.354 g, 0.042 mol) was added over 20 min. The reaction was stirred
target molecular weight) was used as macro-RAFT agent to synthesize
p(BP-b-VP) diblock copolymers. p(BP), 2-vinylpyridine, AIBN,
chlorobenzene, and a stir bar were added to a round-bottom flask
and sealed with a rubber septum. The contents were degassed by
sparging with dry nitrogen for 20 min. The polymerization was
initiated by placing the flask in an oil bath at 70 °C. After 6 h reaction,
the polymerization was terminated by quenching the flask in ice water
and precipitating the polymer in methanol. The polymer was dried
under vacuum.
for an additional 15 min before carbon disulfide (3.042 g, 0.040 mol)
in acetone (4.036 g, 0.069 mol) was added over 20 min, during which
time the color turned red. Ten minutes later, chloroform (7.125 g,
0
.060 mol) was added in one portion, followed by dropwise addition
of 50% sodium hydroxide solution (16 g, 0.2 mol) over 30 min. The
reaction was stirred overnight. 60 mL of water was added, followed by
1
0 mL of concentrated HCl to acidify the aqueous solution. N was
2
purged through the reactor with vigorous stirring to help evaporate off
acetone. The solid was collected with a Buchner funnel and then
stirred in 100 mL of 2-propanol. The undissolved solid was filtered off.
The 2-propanol solution was concentrated to dryness, and the
resulting solid was recrystallized from hexanes to afford 9.25 g of
Surface Modification of HA with p(BP-b-VP) Block Polymer
HA-p(BP-b-VP)). A 5 mg/mL of p(BP-b-VP) solution was prepared
(
by dissolving 50 mg of p(BP-b-VP) in a 10 mL of H CCl . Dried HA
2
2
1
was immersed in the polymer solution with shaking for 24 h. The
modified HA was then added to centrifuge tubes and purified via
centrifugation at ∼8300 rcf to form a pellet, followed by removal of the
yellow crystalline solid. Yield: 63.5%. H NMR: 0.99 (t, 3 H), 1.35−
1
.46 (m, 20 H), 1.76 (s, 6 H), 3.42 (t, 2H), 13.12 (s, 1H). MS (M +
+
Na , chem): calcd: 387.2. Found: 387.2.
Synthesis of Tetraethyl (N,N-Dibenzyl)aminomethylbis-
phosphonate) (TEBAP). Diethyl phosphite (12.8 g, 93 mmol),
supernatant and resuspension with H CCl . This process was repeated
2 2
(
4 times. The pure sample was stored in a dust-free environment at
room temperature until use.
triethyl orthoformate (5.3 g, 35.5 mmol), and dibenzylamine (5.9 g, 30
mmol) were mixed in a 100 mL three-necked round-bottom flask. The
Preparation of p(BP-b-VP) Membrane on Glass Slide (p(BP-
solution was heated at 150 °C under a N atmosphere for 5 h. After
b-VP-G)). The p(BP-b-VP) solution (1 mg/mL) was prepared by
2
cooling to RT, the mixture was dissolved in CHCl (250 mL), and the
dissolution of p(BP-b-VP) in HCCl . The solution was then filtered
3
3
solution was washed with 3 × 60 mL of 5% aqueous NaOH and 2 ×
through a 0.45 μm Teflon syringe filter and cast onto a clean glass
substrate by simply adding drops of the solution to the glass. The
7
5 mL of brine. The organic fraction was dried with anhydrous
B
dx.doi.org/10.1021/ma501997x | Macromolecules XXXX, XXX, XXX−XXX

Macromolecules
Article
Scheme 1. Synthetic Scheme for the Monomer and Block Copolymer
membranes were slowly dried at RT for 12 h and then in vacuo for 12
h. These were indicated as p(BP-b-VP-G).
suspension was taken, diluted appropriately, and plated on L-agar
plates for 20 h incubation. Theoretically, each surviving bacterium
develops into a distinct colony after incubation, and the number of
viable bacteria referred to as colony forming units (CFU), thus
providing a direct measure of bacterial viability. A bacterial suspension
then was sprayed onto a p(BP-b-VP)-Ag film on glass slide or a blank
glass slide (no modification) in a fume hood by using a commercial
chromatography sprayer (VWR Scientific) (spray rate of 10 mL/min).
After drying for 10 min under air, the slide was placed in a Petri dish,
and then growth agar (0.7% agar in a yeast dextrose broth, autoclaved,
and cooled to 37 °C) was added. The Petri dish was closed, sealed,
and incubated at 37 °C overnight. The surface of samples was washed
using DI water to remove the dead and unattached bacteria. Bacteria
remaining on the sample surfaces were stained with fluorescein (Na
salt) according to standard protocol. The bacteria cells on the surface
were imaged using a Nikon Eclipse 80i microscope with 100× lens
through a FITC filter. Photographs were taken with a CCD-Cool
SNAP camera (Roper scientific, Inc.).
Preparation of p(BP-b-VP) Containing Ag Nanoparticles
0
(
p(BP-b-VP-Ag )). The p(BP-b-VP) block copolymer, HA-p(BP-b-
VP), and p(BP-b-VP)-G were added to aqueous AgNO (20 mL, 5.0
3
−
1
mmol L ) solutions for 24 h to allow the pyridine groups to complex
the Ag . The samples were subsequently washed thoroughly with
+
water (200 mL) and stored in DI water for 12 h to remove any excess
AgNO . An excess of aqueous NaBH was then added with vigorous
3
4
stirring, and then the mixture was kept at RT for 12 h. The resultant
Ag nanoparticles containing materials were washed copiously with DI
water, stored in 500 mL of DI water, and dried under vacuum at 80 °C
for 12 h. These samples were stored in a dust-free environment at
room temperature until use. They abbreviations used to indicate Ag
0
nanoparticles were present are p(BP-b-VP)-Ag for p(BP-b-VP) block
0
copolymer with Ag nanoparticles, HA-p(BP-b-VP)-Ag for HA-p(BP-
0
b-VP) with Ag nanoparticles, and p(BP-b-VP)-G-Ag for p(BP-b-VP)-
G with Ag nanoparticles.
Characterization. 1H NMR was collected on a Varian Unity
spectrometer at 400 MHz at 30 °C with tetramethylsilane (TMS) as
the internal standard. Thermogravimetric analyses (TGA) was done
with a PerkinElmer TGA-2 thermogravimetric analyzer (Inspiratech
Control Experiment. HA was soaked in aqueous AgNO (20 mL,
.0 mmol L ) solutions for 24 h. The sample was subsequently
3
−1
5
washed thoroughly with water (200 mL) 6 times and stored in DI
water for 12 h to remove any excess AgNO . An excess of aqueous
3
2
000 Ltd., UK) at a heating rate of 10 °C/min. All the samples were
NaBH was then added with vigorous stirring, and then the mixture
4
first vacuum-dried and kept in the TGA furnace at 150 °C in a
nitrogen atmosphere for 30 min to remove water before TGA
characterization. Transmission electron microscopy (TEM) was
carried out on a Jeol JEM 2000EXII operating at 200 kV. Differential
scanning calorimetric (DSC) experiments were carried out under a
nitrogen atmosphere using a PerkinElmer DSC-7 system at a heating
rate of 10 °C/min. Wide-angle X-ray diffraction (XRD) measurements
were obtained on a Rigaku Max 2500 V PC X-ray diffract meter
was kept at RT for 12 h. The resultant material was washed copiously
with DI water, stored in 500 mL of DI water, and dried under vacuum
at 80 °C for 12 h. The sample was named as HA-C. 0
Ag Leaching Experiment. 0.1 g of p(BP-b-VP)-Ag was soaked in
DI for 2 weeks at room temperature. After filtration, the concentration
of Ag in the filtrate was tested by inductively coupled plasma mass
spectrometer (ICP-MS).
Antimicrobial Activity Assay. Antimicrobial activity of the
generated materials was evaluated using L. plantarum. A colony of L.
plantarum bacteria was cultivated in Luria−Bertani broth (containing
(
8
Japan) with Cu Kα radiation (40 kV, 200 mA) with a scanning rate of
°C/min. X-ray photoelectron spectroscopy (XPS) analysis of the
samples was performed using a Thermo Scientific K-Alpha ESCA
instrument equipped with aluminum Kα1,2 monochromatized radiation
at 1486.6 eV X-ray source. For Ag determination, ICP-MS (Elan DRC
II (PerkinElmer SCIEX, Norwalk, CT)) was used.
1
3
0 g/L peptone, 10 g/L sodium chloride, and 5 g/L yeast extract) at
7 °C, shaking at 160 rpm for 24 h. The bacteria were diluted with 0.1
mol/L phosphate buffer solution (PBS, pH = 7) to the desired
0
concentration. A certain amount of HA and HA-p(BP-b-VP)-Ag were
immersed in the bacterial suspension and then shaken at 37 °C for 20
h. After an overnight culture at 37 °C, colony forming units (CFU) of
L. plantarum was determined (using BioMate 3S spectrophotometer)
from the measured absorbance at 600 nm wavelength using previously
established standard calibration curves. A certain amount of
RESULTS AND DISCUSSION
■
Synthesis. The monomer AABP was synthesized from the
reaction of acryloyl chloride and tetraethyl aminomethylbis-
(phosphonate) (Scheme 1). The latter was prepared by
C
dx.doi.org/10.1021/ma501997x | Macromolecules XXXX, XXX, XXX−XXX

Macromolecules
Article
Figure 4. DSC traces for (red) p(BP), and (black) p(BP-b-VP) under
a nitrogen atmosphere.
1
Figure 1. H NMR of (black) AABP, (red) p(BP), and (blue) p(BP-b-
VP).
Figure 5. XPS spectra in the Ag 3d region for (black) p(BP-b-VP)-Ag+
0
and (red) p(BP-b-VP)-Ag .
Figure 2. 31P NMR of (black) AABP, (red) p(BP), and (blue) p(BP-b-
VP).
+
Figure 6. XRD for (blue) p(BP-b-VP), (red) p(BP-b-VP)-Ag , and
0
(
black) p(BP-b-VP)-Ag .
Figure 3. TGA traces for (black) p(BP) and (red) p(BP-b-VP) under
a nitrogen atmosphere.
reaction of dibenzylamine, triethyl orthoformate, and diethyl
phosphite to give the intermediate TEBAP, followed by
debenzylation with H2 and Pd/C as the catalyst. The
deprotected tetraethylaminomethylbis(phosphonate) was con-
verted quantitatively into monomer AABP by means of a
substitution reaction with an excess of chloroacetyl chloride.
1
31
The AABP structure was confirmed by H and P NMR and
mass spectrometry (MS) (in Experimental Section).
+
Figure 7. TEM micrographs for (left) p(BP-b-VP)-Ag and (right)
p(BP-b-VP)-Ag .
0
Bisphosphonate oligomers were synthesized by RAFT
polymerization in DMF at 70 °C using AABP as the monomer,
RAFT-COOH as the chain transfer agent, and AIBN as the
spectra of AABP and p(BP). Three peaks of AABP at 5.15,
5.71, and 5.30 ppm were assigned to the vinyl group protons.
1
initiator as shown in Scheme 1. Figure 1 shows H NMR
D
dx.doi.org/10.1021/ma501997x | Macromolecules XXXX, XXX, XXX−XXX

Macromolecules
Article
Figure 8. Photographs of agar plates with L. plantarum after exposure
0
+
to (a) p(BP-b-VP)-Ag , (b) p(BP-b-VP)-Ag , and (c) control
experiment.
Figure 12. TEM micrographs of (a) HA, (b) HA-p(BP-b-VP), (c)
HA-p(BP-b-VP)-Ag , and (d) HA-C.
Figure 9. SEM images of p(BP-b-VP) film on glass slides: (a) top
surface; (b) cross section.
0
Figure 13. Photographs of (a, 1−3) L. plantarum solution treated with
0
HA-p(BP-b-VP)-Ag and (a, 4) a blank, (b, 5,6) L. plantarum solution
treated with HA, and (c, 7) L. plantarum solution treated with HA-C.
Images were aquired after 20 h culture at 37 °C.
Figure 10. Fuorescence microscope images of fluorescently stained L.
0
plantarum attached to glass slide with (a) p(BP-b-VP)-Ag and (b)
bare glass after incubation for 20 h.
Figure 11. Photographs of (a) blank glass slide and (b) glass slide
0
coated by p(BP-b-VP)-Ag after 3 days.
After polymerization, these three peaks disappeared and a new
peak in 0.92 ppm appeared which was attributed to the proton
in the end methyl group of the RAFT-COOH reagent. The
number of repeat units of bisphosphonate can be calculated by
Figure 14. CFUs mL− of L. plantarum after 20 h of growth.
1
the integration ratio of the peak at 4.22 ppm (O−CH −) and
to P of phosphonate. The small difference comes from the
different P chemical environment in monomer and polymer.
Subsequently, the p(BP) oligomer was used as a macro-
chain-transfer agent, and 2-vinylpyridine was selected as
monomer, for the synthesis of p(BP-b-VP) block copolymers.
After the polymerization, the polymer was precipitated into
2
0
.92 ppm (CH − in RAFT-COOH). The calculated repeated
3
1
units of p(BP) by H NMR was 28, which is close to the repeat
3
1
unit target (30). As shown in Figure 2, P NMR spectra of
AABP and p(BP) exhibit similar chemical shifts around 16.1
ppm for AABP and 17.0 ppm for p(BP) which were attributed
E
dx.doi.org/10.1021/ma501997x | Macromolecules XXXX, XXX, XXX−XXX

Macromolecules
Article
0
Figure 15. Photographs of samples of L. plantarum after exposure to HA-p(BP-b-VP)-Ag (a: 0.5%; b: 0.75%; c: 1%). (d, e) HA (d: 0.5%; e: 1%)
treated solution. (f) HA-C (1%) treated solution. (g) Blank solution.
water to remove solvent, unreacted p(BP), and 2-vinylpyridine.
p(BP-b-VP) is at 92 °C, which can be assigned to the presence
1
As shown in Figure 1, compared with H NMR of p(BP), four
of strong polar pyridine oligomer.
1
0
new peaks in H NMR of p(BP-b-VP) appear between 6.31 and
p(BP-b-VP)-Ag . Since we showed that p(BP-b-VP) could
8
.30 ppm that were attributed to the pyridine protons. 100
be synthesized, we went on to develop it as an antibacterial
1
polymer and ultimately a coating. To accomplish this, the
pyridine repeated units were added, as determined by H NMR
integration ratio calculation of the peak at 6.31 ppm to that at
+
polymer was exposed to Ag , which was complexed by the
+
pyridine groups. The Ag is subsequently reduced by exposure
4
.22 ppm. These two polymers exhibited similar chemical shifts
31
to NaBH to obtain Ag nanoparticles. This is shown in Scheme
of P NMR at 17.0 ppm, which also approve successful
4
+
1
. ICP-MS analysis confirmed that the amount of Ag that was
loaded into p(BP-b-VP) is 29.8 wt %. The Ag containing
polymer was named as p(BP-b-VP)-Ag . ICP-MS analysis also
synthesis of p(BP-b-VP), as shown in Figure 2.
Thermal Properties. The thermal stabilities of the p(BP)
and p(BP-b-VP) were analyzed by TGA under flowing nitrogen
+
+
revealed that the Ag content was ∼25.7% after the NaBH
4
(Figure 3). These samples were preheated to 150 °C and held
reduction process. XPS was also used to confirm the presence
of Ag in the polymer after reduction by analyzing the Ag 3d3/2
and Ag 3d5/2 regions, as these regions are very sensitive to the
chemical environment surrounding the Ag and can provide
important information to distinguish between Ag ions and
isothermally for 30 min for moisture removal. The initial weight
loss of p(BP) and p(BP-b-VP) was observed at 250 °C and was
21
assigned to the decomposition of the phosphonate group.
The initial 5% weight loss temperature of p(BP) ranged from
2
2
50 to 264 °C, which is lower than that of p(BP-b-VP) (from
50 to 285 °C). The residue of p(BP-b-VP) at 600 °C
11a
metallic Ag. This data is shown in Figure 5, which reveals the
+
Ag 3d3/2 at 373.9 eV and Ag 3d5/2 367.9 eV for Ag . These
remained 17%, which was attributed to the carbonization of
pyridine groups. Pyridine oligomer increased thermal stability
of polymer. From the thermal testing, it can be concluded that
the p(BP-b-VP) would meet the thermal stability requirements
for antibacterial surface coating applications.
In order to further investigate the basic properties of p(BP-b-
VP), the glass transition temperature was characterized by
DSC. For comparison, the thermal property of p(BP) was also
tested and shown in Figure 4. In order to eliminate the thermal
history, the DSC curves of the second scan were recorded. No
melting point was observed for the two polymers, which means
peaks shift to 373.6 and 367.6 eV through the reduction
process, which is characteristic of Ag 3d3 and 3d5/2 Ag
/2
22
nanoparticle, respectively.
+
XRD was also conducted on p(BP-b-VP), p(BP-b-VP)-Ag ,
0
and p(BP-b-VP)-Ag , and the data are shown in Figure 6. The
+
curves of p(BP-b-VP) and p(BP-b-VP)-Ag were broad and
without obvious peak features, which also indicated that they
0
were all amorphous. The p(BP-b-VP)-Ag displayed a new peak
at 2θ = 38.4° which is the characteristic (1, 1, 1) plane of face-
23
centered cubic crystalline Ag. This confirms the formation of
+
crystalline Ag in p(BP-b-VP) matrix by the reduction of Ag .
+
they are amorphous. The glass transition temperatures (T ) of
Figure 7 shows TEM micrographs of p(BP-b-VP)-Ag and
g
F
dx.doi.org/10.1021/ma501997x | Macromolecules XXXX, XXX, XXX−XXX

Macromolecules
Article
0
0
p(BP-b-VP)-Ag . The TEM image of p(BP-b-VP)-Ag revealed
that this sample is constituted by well-dispersed Ag nano-
particles with diameters of 5−15 nm. In order to investigate
reduced into Ag nanoparticles by exposure to NaBH . Figure
4
12c illustrates the TEM image of HA-p(BP-b-VP)-Ag nano-
particles. Ag nanoparticles with the diameters of 5−15 nm
deposited on the surfaces of HA particles. The well-dispersed
distribution of Ag nanoparticles indicates that the aggregation
problem of Ag nanoparticles has been avoided, which can result
in large surface area and great antibacterial property. In order to
evaluate the function of polymer in the process, sample HA-C
0
leaching of Ag from p(BP-b-VP)-Ag , the polymer was stored in
DI water for 2 weeks, and the solution was analyzed by ICP-
MS. Very low Ag leaching was detected (0.3 ppm), indicating
0
that the Ag nanoparticles were stable in p(BP-b-VP)-Ag .
The antibacterial properties of p(BP-b-VP)-Ag0 were
determined by the standard disk diffusion assay according to
the procedure on Luria−Bertani agar medium reported by
+
was prepared by exposing HA to Ag without previous p(BP-b-
VP) exposure. The TEM image of HA-C is shown in Figure
12d. As can be seen, no Ag nanoparticles were observed on the
HA, indicating that p(BP-b-VP) played a key role in generating
Ag nanoparticles on the HA to render them antibacterial.
2
4
Elgayyar et al. In these tests L. plantarum was used as model
bacteria. L. plantarum is a Gram-positive bacterium that is
found in dairy, meat, many vegetable fermentations, and the
mouth, which can break down sugars as part of their
metabolism, forming lactic acid and promoting tooth (main
0
A certain amount of HA-p(BP-b-VP)-Ag was added to L.
plantarum solution in yeast/dextrose broth. The suspension
was shaken for 10 min and then incubated at 37 °C for 20 h.
For comparison purposes, the unmodified HA, HA-C, and
blank experiment (nothing added to bacteria solution) were
also conducted in the same conditions. Figure 13 displays the
optical images of these samples after 20 h culture at 37 °C. The
solution of HA, HA-C, and blank become cloudy, resulting
from L. plantarum growth. All of HA- p(BP-b-VP)-Ag
nanoparticles treated solution remained clear and transparent,
which indicated the growth of L. plantarum was inhibited. The
concentration of L. plantarum was tested by absorbance
measurement at 600 nm wavelength, and the results are
shown in Figure 14. The concentration of L. plantarum solution
treated by HA-p(BP-b-VP)-Ag nanoparticles ranged from 1.99
25
mineral component: HA) decay. All bactericidal activity tests
were performed on tests disks of 11 mm diameter and 0.3 mm
thickness. The disk of p(BP-b-VP)-Ag was very efficient as an
antibacterial agent, which can create a large zone of inhibition
in the bacterial growth lawn, as shown in Figure 8. The disk of
p(BP-b-VP) and blank (just L. plantarum was added in the
dish) were also tested as controls. The growth bacteria filled all
of these disc areas and covered the p(BP-b-VP) surface, with no
inhibition zone forming, which implies that these discs have no
antibacterial activity.
0
0
p(BP-b-VP)-Ag on Glass Slide and Antibacterial
Properties. In order to investigate this system’s practical
application as an antibacterial coating, a compact and smooth
p(BP-b-VP) film with thickness of 2.5 μm was prepared on a
8
8
−1
× 10 to 2.36 × 10 CFU mL with the HA-p(BP-b-VP)-Ag
26
glass slide using a solution casting method, as shown in Figure
. Then, Ag nanoparticles were incorporated into film. The
content increasing from 0.5% to 1.0%. These values are close to
8
9
the original L. plantarum concentration (1.71 × 10 CFU
0
−1
p(BP-b-VP)-Ag film was used for antibacterial test with a blank
mL ). The concentration of L. plantarum solution treated by
8
−1
6
HA, HA-C, and blank increased from 1.71 × 10 CFU mL to
.85 × 10 , 1.74 × 10 , and 1.86 × 10 CFU mL , respectively,
slide used as control. A suspension of L. plantarum (1 × 10
9
9
9
−1
−1
1
CFU mL ) was sprayed onto the respective modified and
unmodified glass slides using a commercial chromatography
sprayer (VWR Scientific) (spray rate of 5 mL/min). After being
air-dried, the slides were placed in Petri dishes, and then growth
agar (0.7% agar in a yeast/dextrose broth) was added. The Petri
dishes were sealed and incubated for 20 h at 37 °C. Then, the
which are almost 10 times larger than these of HA-p(BP-b-VP)-
Ag nanoparticles treated solution (Figure 14). These indicated
that the HA- p(BP-b-VP)-Ag nanoparticles exhibited excellent
antibacterial property. In order to investigate the L. plantarum
activity after treated by HA and HA-p(BP-b-VP)-Ag nano-
particles, the L. plantarum solutions with same concentration (2
slides were taken out, washed with H O to remove agar and
2
6
−1
×
10 CFU mL ) were prepared from above samples. 0.1 mL
unattached L. plantarum, and exposed to fluorescein sodium
salt (according to standard protocol). Figure 10 shows
representative microscope images of the fluorescently stained0
L. plantarum attached to the surfaces of the p(BP-b-VP)-Ag
modified glass slide and the control. A high bacteria density at
the glass slide surface was observed, while no bacteria was
observed on the surface of p(BP-b-VP)-Ag film; this clearly
shows the antibacterial properties of the p(BP-b-VP)-Ag film.
After being incubated for 3 days, several visible bacterial
colonies appeared on glass slide surface, as shown in Figure 11,
while no bacterial colonies were observed on the p(BP-b-VP)-
of L. plantarum solution was painted on the surface of nutrient
agar media, and then samples were incubated for 20 h at 37 °C.
The result is shown in Figure 15. In this manner, clear
nongrowth L. plantarum was observed in the agar experiments
from the solution treated by HA-p(BP-b-VP)-Ag nanoparticles,
which indicated that the L. plantarum was inactivated. On the
other hand, L. plantarum colonies covered all of agar surface
when HA and HA-C treated and blank L. plantarum was used.
0
0
CONCLUSION
In this submission we synthesized a novel block copolymer that
was capable of adhering to HA surfaces as well as complexing
■
0
Ag film.
HA Containing p(BP-b-VP)-Ag0 and Antibacterial
Properties. Next, we wanted to prove that the p(BP-b-VP)-
Ag could be deposited on HA surfaces. To accomplish this,
HA particles were immersed into p(BP-b-VP) solution at room
temperature and then washed three times by fresh H CCl to
remove unattached polymer. TEM analysis revealed that a thin
and uniform polymer layer (∼4 nm) was formed around the
HA due to strong interaction of p(BP-b-VP) and HA, as shown
in Figure 12. Then, the substrate (HA- p(BP-b-VP)) was
immersed into dilute AgNO solution, whereupon Ag ions were
bound to the polymer coating on HA. Finally, Ag ions were
+
Ag . The polymer was characterized by NMR, TGA, XRD,
0
XPS, TEM, and microscopy. The purpose of synthesizing such
a polymer was to allow HA-containing medical devices,
implants, and teeth to be coated with the polymer, while the
2
2
+
0
Ag could be reduced to Ag , allowing the surfaces to be
rendered antibacterial. Through our processing, we were able to
modify HA surfaces with polymer layers of ∼4 nm and Ag
nanoparticles of 5−15 nm in diameter. We showed that the
polymer and the polymer-coated surfaces (with Ag nano-
particles) were able to kill the bacteria L. plantarum very
3
G
dx.doi.org/10.1021/ma501997x | Macromolecules XXXX, XXX, XXX−XXX

Macromolecules
Article
effectively. We feel that these polymers have promise for future
use as antibacterial coatings for a wide variety of HA surfaces,
while the concept can be adapted for a variety of other
materials.
(12) (a) Yuan, W.; Wei, J.; Lu, H.; Fan, L.; Du, J. Chem. Commun.
2
012, 48, 6857−6859. (b) Sun, T.; Wang, G.; Feng, L.; Liu, B.; Ma, Y.;
Jiang, L.; Zhu, D. Angew. Chem., Int. Ed. 2004, 43, 357−360.
c) Sileika, T. S.; Barrett, D. G.; Zhang, R.; Lau, K. H. A.; Messersmith,
(
P. B. Angew. Chem., Int. Ed. 2013, 52, 10766−10770.
13) Fan, Z.; Liu, B.; Wang, J.; Zhang, S.; Lin, Q.; Gong, P.; Ma, L.;
Yang, S. Adv. Funct. Mater. 2014, 24, 3933−3943.
14) Taglietti, A.; Arciola, C. R.; D’Agostino, A.; Dacarro, G.;
(
AUTHOR INFORMATION
■
Corresponding Author
(
*E-mail michael.serpe@ualberta.ca (M.J.S.).
Montanaro, L.; Campoccia, D.; Cucca, L.; Vercellino, M.; Poggi, A.;
Pallavicini, P.; Visai, L. Biomaterials 2014, 35, 1779−1788.
Notes
(15) (a) Agarwal, A.; Weis, T. L.; Schurr, M. J.; Faith, N. G.;
The authors declare no competing financial interest.
Czuprynski, C. J.; McAnulty, J. F.; Murphy, C. J.; Abbott, N. L.
Biomaterials 2010, 31, 680−690. (b) Guo, L.; Yuan, W.; Lu, Z.; Li, C.
M. Colloids Surf., A 2013, 439, 69−83.
ACKNOWLEDGMENTS
M.J.S. acknowledges funding from the University of Alberta
the Department of Chemistry and the Faculty of Science), the
■
(
(16) (a) Fei, X.; Jia, M.; Du, X.; Yang, Y.; Zhang, R.; Shao, Z.; Zhao,
X.; Chen, X. Biomacromolecules 2013, 14, 4483−4488. (b) Chen, M.;
Zhao, Y.; Yang, W.; Yin, M. Langmuir 2013, 29, 16018−16024.
(c) Reithofer, M. R.; Lakshmanan, A.; Ping, A. T.; Chin, J. M.; Hauser,
C. A. Biomaterials 2014, 35, 7535−7542. (d) Epa, V.; Hook, A.;
Chang, C.; Yang, J.; Langer, R.; Anderson, D.; Williams, P.; Davies, M.;
Alexander, M.; Winkler, D. Adv. Funct. Mater. 2014, 24, 2085−2093.
Natural Sciences and Engineering Research Council of Canada
NSERC), the Canada Foundation for Innovation (CFI), and
(
the Alberta Advanced Education & Technology Small Equip-
ment Grants Program (AET/SEGP).
(
17) (a) Hoang, Q. Q.; Sicheri, F.; Howard, A. J.; Yang, D. S. Nature
003, 425, 977−980. (b) Yamagishi, K.; Onuma, K.; Suzuki, T.;
Okada, F.; Tagami, J.; Otsuki, M.; Senawangse, P. Nature 2005, 433,
19−819.
18) Zhang, S.; Gangal, G.; Uludag
07−531.
19) (a) Senaratne, S. G.; Colston, K. W. Breast Cancer Res. 2001, 4,
8. (b) Fournier, P.; Boissier, S.; Filleur, S.; Guglielmi, J.; Cabon, F.;
Colombel, M.; Clezardin, P. Cancer Res. 2002, 62, 6538−6544.
20) Schultz, M.; Veltkamp, C.; Dieleman, L. A.; Grenther, W. B.;
Wyrick, P. B.; Tonkonogy, S. L.; Sartor, R. B. Inflamm. Bowel Dis.
002, 8, 71−80.
21) Price, D.; Cunliffe, L. K.; Bullett, K. J.; Hull, T. R.; Milnes, G. J.;
Ebdon, J. R.; Hunt, B. J.; Joseph, P. Polym. Degrad. Stab. 2007, 92,
101−1114.
22) Thiel, J.; Pakstis, L.; Buzby, S.; Raffi, M.; Ni, C.; Pochan, D. E. J.;
Shah, S. I. Small 2007, 3, 799−803.
23) Southward, R. E.; Thompson, D. W.; St. Clair, A. K. Chem.
Mater. 1997, 9, 501−510.
24) Elgayyar, M.; Draughon, F.; Golden, D.; Mount, J. J. Food Prot.
001, 64, 1019−1024.
25) Huang, J.; Wong, H.-L.; Zhou, Y.; Wu, X.; Grad, H.;
Komorowski, R.; Friedman, S. J. Controlled Release 2000, 67, 293−307.
26) Zhang, Q.; Zhang, Q.; Wang, J.; Zhang, S.; Li, S. Polymer 2010,
1 (23), 5407−5416.
REFERENCES
■
2
(
2
1) (a) Neoh, K. G.; Hu, X.; Zheng, D.; Kang, E. T. Biomaterials
012, 33, 2813−2822. (b) Wang, B.; REN, K.; Chang, H.; Wang, J.; Ji,
8
J. ACS Appl. Mater. Interfaces 2013, 34, 4136−4143.
(
̆
, H. Chem. Soc. Rev. 2007, 36,
(
2) (a) Donlan, R. M.; Costerton, J. W. Clin. Microbiol. Rev. 2002, 15,
5
(
1
1
2
67−193. (b) Costerton, J.; Stewart, P. S.; Greenberg, E. Science 1999,
84, 1318−1322. (c) McLean, R. J.; Nickel, J. C.; Cheng, K.-J.;
Costerton, J. W.; Banwell, J. G. Crit. Rev. Microbiol. 1988, 16, 37−79.
(
́
3) (a) Engemann, J. J.; Carmeli, Y.; Cosgrove, S. E.; Fowler, V. G.;
(
Bronstein, M. Z.; Trivette, S. L.; Briggs, J. P.; Sexton, D. J.; Kaye, K. S.
Clin. Infect. Dis. 2003, 36, 592−598. (b) Zimmerli, W.; Ochsner, P.
Infection 2003, 31, 99−108.
2
(
(
4) O’Driscoll, S. W.; Fitzsimmons, J. S. The role of periosteum in
cartilage repair. Clin. Orthop. Relat. Res. 2001, 391, S190−S207.
1
(
(
5) Smeeth, L.; Thomas, S. L.; Hall, A. J.; Hubbard, R.; Farrington,
P.; Vallance, P. Risk of myocardial infarction and stroke after acute
infection or vaccination. N. Engl. J. Med. 2004, 351, 2611−2618.
(
(
6) (a) Banerjee, I.; Pangule, R. C.; Kane, R. S. Adv. Mater. 2011, 23,
6
90−718. (b) Li, M.; Neoh, K. G.; Kang, E. T.; Lau, T.; Chiong, E.
(
2
(
Adv. Funct. Mater. 2014, 24, 1631−1643. (c) Yang, W. J.; Cai, T.;
Neoh, K.-G.; Kang, E.-T.; Teo, S. L.-M.; Rittschof, D. Biomacromo-
lecules 2013, 14, 2041−2051. (d) Venault, A.; Yang, H.-S.; Chiang, Y.-
C.; Lee, B.-S.; Ruaan, R.-C.; Chang, Y. ACS Appl. Mater. Interfaces
(
5
2
014, 6, 3201−3210. (e) Ng, V. W. L.; Tan, J. P. K.; Leong, J.; Voo, Z.
X.; Hedrick, J. L.; Yang, Y. Y. Macromolecules 2014, 47, 1285−1291.
f) García-Fernandez, L.; Cui, J.; Serrano, C.; Shafiq, Z.; Gropeanu, R.
(
́
A.; Miguel, V. S.; Ramos, J. I.; Wang, M.; Auernhammer, G. K.; Ritz, S.
Adv. Mater. 2013, 25, 529−533.
(
7) Sileika, T. S.; Kim, H.-D.; Maniak, P.; Messersmith, P. B.
Antibacterial performance of polydopamine-modified polymer surfaces
containing passive and active components. ACS Appl. Mater. Interfaces
2
011, 3, 4602−4610.
8) Lienkamp, K.; Tew, G. N. Chem.Eur. J. 2009, 15, 11784−
1800.
9) Gurumurthy, M.; Rao, M.; Mukherjee, T.; Rao, S. P. S.; Boshoff,
H. I.; Dick, T.; Barry, C. E.; Manjunatha, U. H. Mol. Microbiol. 2013,
7, 744−755.
10) (a) Liu, Y.; Ma, W.; Liu, W.; Li, C.; Liu, Y.; Jiang, X.; Tang, Z. J.
(
1
(
8
(
Mater. Chem. 2011, 21, 19214−19218. (b) Hu, R.; Li, G.; Jiang, Y.;
Zhang, Y.; Zou, J.-J.; Wang, L.; Zhang, X. Langmuir 2013, 29, 3773−
3
779.
(
11) (a) Shah, M. S. A. S.; Nag, M.; Kalagara, T.; Singh, S.;
Manorama, S. V. Chem. Mater. 2008, 20, 2455−2460. (b) Gutman, O.;
Natan, M.; Banin, E.; Margel, S. Biomaterials 2014, 35, 5079−5087.
(
c) Park, J.; Kim, J.; Singha, K.; Han, D.-K.; Park, H.; Kim, W. J.
Biomaterials 2013, 34, 8766−8775. (d) Jin, G.; Qin, H.; Cao, H.; Qian,
S.; Zhao, Y.; Peng, X.; Zhang, X.; Liu, X.; Chu, P. K. Biomaterials 2014,
3
5, 7699−7713.
H
dx.doi.org/10.1021/ma501997x | Macromolecules XXXX, XXX, XXX−XXX